This invention is concerned with novel HIV protease inhibitors, a process for their manufacture, pharmaceutical compositions and the use of such compounds in medicine. In particular, the compounds are peptide mimetics which act as inhibitors of the HIV aspartyl protease, an essential enzyme in the replicative life cycle of HIV. Consequently, the compounds of this invention may be advantageously used in the treatment of HIV infection, either alone or in combination with other inhibitors of HIV viral replication or with pharmacoenhancers such as cytochrome P450 inhibitors.
The human immunodeficiency virus HIV is the causative agent of acquired immunodeficiency syndrome (AIDS), a disease characterised by the destruction of the immune system, particularly of the CD4+ T-cell, with attendant susceptibility to opportunistic infections. HIV infection is also associated with a precursor AIDs-related complex (ARC), a syndrome characterised by symptoms such as persistent generalised lymphadenopathy, fever and weight loss.
In common with other retroviruses, the HIV genome encodes protein precursors known as gag and gag-pol which are processed by the viral protease to afford the protease, reverse transcriptase (RT), endonuclease/integrase and mature structural proteins of the virus core. Interruption of this processing prevents the production of normally infectious virus. Considerable efforts have been directed towards the control of HIV by inhibition of virally encoded enzymes. In particular, much effort has been directed towards the inhibition of HIV protease, and the HIV protease inhibitors (PIs) saquinavir, ritonavir, nelfinavir, indinavir, amprenavir and lopinavir have been approved for treatment of HIV infections. Because of the emergence of resistant virus during monotherapy, current clinical practice is to use such protease inhibitors in combination therapy, typically with RT inhibitors.
The emergence of resistant virus can be attributed to errors introduced by the HIV reverse transcriptase, in conjunction with a high virus replication rate. It is likely that mutations that lead to resistant virus occur spontaneously but remain undetectable until initiation of therapy leads to a selective pressure for the emergence of virus with replicative advantage over the wildtype population. In the context of HIV protease inhibition, accumulation of mutations that lead to a reduction in inhibitor binding while maintaining sufficient substrate turnover can lead to drug resistance. Although the onset of drug resistance can be delayed to some extent by the use of combinations of drugs, there remains a need for more effective HIV protease inhibitors that retain activity against PI-resistant and multi-PI resistant viruses.
In one aspect of the present invention, there are provided novel compounds which are potent inhibitors of the HIV aspartyl protease and which accordingly show a potential to be efficacious in the treatment of HIV related diseases. Compounds of the invention may also therefore show the potential to inhibit the replication of virus that is resistant to commonly used protease inhibitors.
The compounds of the present invention may be characterized by the following formula I 
as individual isomers, racemates, non-racemic mixtures or mixtures of diastereoisomers; wherein n, R1 and R4 are as described below.
The present invention is also directed to pharmaceutical compositions containing compounds of formula I and the use of the compounds of formula I in the treatment or therapy of HIV mediated diseases.
The present invention is directed to, inter alia, a compound of formula I, 
wherein n is 0, 1 or 2;
R1 is naphthyl, quinolinyl or phenyl, optionally substituted by halogen;
R4 is (C1-C7)-alkyl;
A is a group 
xe2x80x83wherein
R2 is hydrogen or (C1-C7) lower alkoxy; and
R3 is (C1-C7)-alkyl;
or pharmaceutically acceptable salts thereof.
The term (C1-C7)-alkyl defines an optionally substituted straight or branched alkyl chain carrying 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms. Alkyl preferably stands for methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and t-butyl.
The term (C1-C7)-alkoxy defines an optionally substituted straight or branched alkoxy chain carrying 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms. Alkoxy preferably stands for methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy and t-butoxy.
The term halogen stands for fluorine, chlorine, bromine and iodine.
The term xe2x80x9cracematexe2x80x9d defines a mixture of 50:50 of pure dextrorotatory and levorotatory enantiomers. The term xe2x80x9cnon racemicxe2x80x9d defines mixtures containing pure dextrorotatory and levorotatory enantiomer at ratios different from 50:50 and varying between 1:99 and 99:1.
Compounds of formula (I) which are acidic can form pharmaceutically acceptable salts with bases such as alkali metal hydroxides, e.g. sodium hydroxide, potassium hydroxide and the like; alkaline earth metal hydroxides, e.g. calcium hydroxide, barium hydroxide, magnesium hydroxide and the like; with organic bases e.g. N-ethyl piperidine, dibenzylamine and the like. Those compounds of formula (I) which are basic can form pharmaceutically acceptable salts with inorganic acids, e.g. hydrohalic acids such as hydrochloric acid and hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid and the like; and with organic acids, e.g. acetic acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malic acid, salicylic acid, citric acid, methanesulphonic acid, p-toluene sulphonic acid and the like. The formation and isolation of such salts can be carried out according to methods known in the art.
Preferred compounds of formula I include the following cyclopentanecarboxylic acid tert-butylamides:
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalen-2-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(quinolin-8-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalen-2-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{3-[3-(4-Fluoro-phenylsulfanyl)-2-methanesulfonylamino-propionylamino]-2-hydroxy-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{3-[3-(4-Fluoro-phenylsulfanyl)-2-methanesulfonylamino-propionylamino]-2-hydroxy-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-2-sulfinyl)-propionylamino]-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-2-sulfonyl)-propionylamino]-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-2-sulfonyl)-propionylamino]-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(quinoline-8-sulfonyl)-propionylamino]-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide;
2-{3-[3-(4-Fluoro-benzenesulfonyl)-2-methanesulfonylamino-propionylamino]-2-hydroxy-4-phenyl-butyl}-4-methyl-cyclopentanecarboxylic acid tert-butylamide.
Further preferred compounds of formula I include the following cyclohexanecarboxylic acid tert-butylamides:
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalen-1-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
N-tert-Butyl-2-[2-hydroxy-3-[[N2-(methanesulfonyl)-S-(2-naphthyl)-D-cysteinyl]amino]-4-phenylbutyl]-4-methoxy-1-cyclohexanecarboxamide;
2-{3-[3-(4-Fluoro-phenylsulfanyl)-2-methanesulfonylamino-propionylamino]-2-hydroxy-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalen-1-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalen-2-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(quinolin-8-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxyl acid tert-butylamide;
2-{3-[3-(4-Fluoro-phenylsulfanyl)-2-methanesulfonylamino-propionylamino]-2-hydroxy-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalen-1-ylsulfanyl)-propionylamino]-4-phenyl-butyl}-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalen-1-ylsulfinyl)-propionylamino]-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-2-sulfinyl)-propionylamino]-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{3-[3-(4-Fluoro-benzenesulfinyl)-2-methanesulfonylamino-propionylamino]-2-hydroxy-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-1-sulfinyl)-propionylamino]-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-2-sulfinyl)-propionylamino]-4-phenyl-butyl-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-1-sulfinyl)-propionylamino]-4-phenyl-butyl}-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-1-sulfonyl)-propionylamino]-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
N-tert-Butyl-2-[2-hydroxy-3-[[N2-(methanesulfonyl)-S-(2-naphthyl)-D-cysteinyl]amino]-4-phenylbutyl]-4-methoxy-1-cyclohexanecarboxamide S,S-dioxide;
2-{3-[3-(4-Fluoro-benzenesulfonyl)-2-methanesulfonylamino-propionylamino-]2-hydroxy-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-1-sulfonyl)-propionylamino]-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-2-sulfonyl)-propionylamino]-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(quinoline-8-sulfonyl)-propionylamino]-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{3-[3-(4-Fluoro-benzenesulfonyl)-2-methanesulfonylamino-propionylaminol]-2-hydroxy-4-phenyl-butyl}-5-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(naphthalene-1-sulfonyl)-propionylamino]-4-phenyl-butyl}-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(quinoline-8-sulfonyl)-propionylamino]-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide;
2-{2-Hydroxy-3-[2-methanesulfonylamino-3-(quinoline-2-sulfonyl)-propionylamino]-4-phenyl-butyl}-4-methoxy-cyclohexanecarboxylic acid tert-butylamide.
Preferred compounds of the invention are compounds of formula 1 wherein
n is 1 or 2
R1 is naphthyl, quinolinyl or phenyl
R4 is methyl
A is a group A1 or A2 wherein
R2 is hydrogen or methoxy
R3 is (C1-C4)-alkyl.
Further preferred compounds of the invention are compounds of formula 1 wherein
n is 1 or 2
R1 is naphthyl,
R4 is methyl
A is a group A1 or A2 wherein
R2 is hydrogen or methoxy
R3 is (C1-C4)-alkyl.
Still further preferred compounds of the invention are compounds of formula 1 wherein
n is 2
R1 is naphthyl,
R4 is methyl
A is a group A1 or A2 wherein
R2 is methoxy
R3 is methyl.
The compounds provided by the present invention, or prodrugs thereof, are potent inhibitors of the HIV aspartyl protease, an essential enzyme in the replicative cycle of the HIV virus. They accordingly are therapeutically active substances in the treatment of HIV-mediated diseases and therefore can be used as medicaments, either alone or combined with other therapeutically active agents.
The compounds provided by the present invention are, in particular, useful in combating HIV disease states such as AIDS.
Any functional (i.e. reactive) group present in a side-chain may be protected, with the protecting group being a group which is known per se, for example, as described in xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd Ed., T. W. Greene and P. G. M. Wuts, John Wiley and Sons, New York, N.Y., 1991. For example, an amino group can be protected by a tert-butoxycarbonyl (BOC), formyl, trityl, benzyloxycarbonyl (Z or Cbz), 9-fluorenylmethyloxcarbonyl (FMOC), trifluoroacetyl, 2-(biphenylyl)isopropoxycarbonyl or isobornyloxycarbonyl group or in the form of a phthalimido group; or a hydroxyl group can be protected by a tert-butyldimethylsilyl, tetrahydropyranyl, 4-methoxybenzyl, or benzyl or acetate etc.; or a carboxyl group can be protected in the form of an ester, for example as a methyl or benzyl or tert-butyl ester. The protecting group may be retained in the final compound or optionally removed by techniques known in the art.
The compounds of general formula I and their pharmaceutically acceptable salts can be manufactured according to the routes depicted in the following schemes. The intermediates, as well as the final compounds falling within the scope of the present invention, may be obtained as mixtures of enantiomers and/or diasteromers which have not been separated further.
While the group BOC is used here below for elucidating the reaction pathways, any other suitable amino protecting group (e.g. those listed above) can be alternatively used. 
According to scheme A, a compound of general formula Ia, b or c can be obtained from a compound of general formula II. Compound II can be deprotected in the presence of a strong acid such as trifluoroacetic acid in e.g. tetrahydrofuran or dichloromethane, after addition of water, and at a temperature varying between 0xc2x0 C. and room temperature (r.t., 25xc2x0 C.). Other suitable strong acids are hydrogen chloride in e.g. water, ethyl acetate or diethylether.
The amine of general formula III thus obtained can be coupled with the appropriate BOC-protected D-amino acid (the synthesis of which is depicted in scheme I) after activating the latter by any conventional method employed to generate peptide bonds. This includes for example the formation of the corresponding hydroxybenzotriazole ester in the presence of a non-nucleophilic base such as triethylamine, pyridine or N-ethyl morpholine.
This reaction can be carried out in a solvent such as tetrahydrofuran or dichloromethane, optionally in the presence of a catalytic quantity of 4-(dimethylamino)pyridine. For solubilisation purposes, the reaction may take place at a temperature between xe2x88x9220xc2x0 C. and 50xc2x0 C. and, more preferably, between 0xc2x0 C. and room temperature.
The removal of the BOC protecting group from the intermediate of general formula IV may take place according to the same procedure applied for deprotecting the compound of general formula II. The corresponding sulphonamide of general formula Ia can be then obtained by reacting the amine of formula V with a sulfonic anhydride or sulfonyl chloride according to methods described in the art, for example by reacting methanesulfonic anhydride (or methanesulphonyl chloride) in a solvent such as tetrahydrofuran, pyridine or dichloromethane, and at a temperature ranging from xe2x88x9240xc2x0 C. to 100xc2x0 C., preferably from 0xc2x0 C. to room temperature in the presence of an organic base such as N-ethyl morpholine.
The compound of general formula Ia can be oxidised to the corresponding sulfoxide of formula Ib or to the corresponding sulfone of general formula Ic, by treatment with an oxidising agent (e.g. metachloroperbenzoic acid) in an organic solvent such as dichloromethane, tetrahydrofuran or methanol, and at a temperature ranging from xe2x88x9220xc2x0 C. to 50xc2x0 C., preferably from 0xc2x0 C. to room temperature.
The compound of general formula IIi, that is the compound of general formula II Wherein the core of A is a 4-methylcyclopentane, can be obtained from compound VIi according to scheme B. 
Compounds of formula II in which A is A2 and R3 is C1-7 other than methyl can be prepared from the intermediate VII using methods described in the art and according to scheme Ba. For example oxidative cleavage of the double bond in VII using for example oxone in dichloromethane/methanol, followed by reductive desulfonylation of the ketosulfone with for example buffered sodium amalgam in methanol or samarium diiodide in tetrahydrofuran gives the ketone VIIi. Elaboration of the ketone to alkenes VIIiii and VIIvii can be achieved by one of a number of methods described in the art for the formation of carbon-carbon bonds through intermediates VIIi or VIIv. VIIv is available from VIIi using the methods described above. For example reaction of VIIi with Wittig and related carbon-carbon double bond forming reagents affords exocyclic alkenes VIIi, while reaction of VIIv with Grignard and related carbon-carbon bond forming reagents followed by acid catalysed elimination afford mixtures of exocyclic and endocyclic alkenes VIIii and VIIvii, both of which can be hydrogenated to give the corresponding 4-substituted cyclopentanes VIIiv. Cyclopentanes VIIiv can in turn be elaborated to the final products in a manner analogous to that described for compound Iii in Scheme B. 
The synthesis of sulfone XVI used for the synthesis of adducts VII is described in scheme C. 
Compound IIii, that is the compound of general formula II wherein the core of A is a 5-methoxycyclohexyl group, can be obtained according to the route described in scheme D, starting from compound X. 
Compound IIiii, that is the compound of general formula II wherein the core of A is a 4-methoxycyclohexyl group, can be obtained from intermediate XXIII according to the route depicted in scheme E. 
Compounds of general formula I in which A is A1 and R2 is lower alkoxy can be prepared by analogous methods to those described in schemes D and E, by alkylation of compounds XXII or XXVII with C2-C7 alkyl halides or related reagents described in the art for the formation of alkyl ethers. For example see J. March (1992) xe2x80x9cAdvanced Organic Chemistry: Reactions, Mechanisms and Structurexe2x80x9d, 4th ed. John Wiley and Sons.
Compound IIiv, that is the compound of general formula II wherein the core of A is a cyclohexyl group, can be obtained according to the route depicted in scheme F: 
Compounds VIi and VIiv (scheme B and F) can be obtained from protected L-phenylalanine as shown in scheme G: 
Compound XXIII (scheme E) can be obtained from intermediate XXXV and commercially available oxazolidinone XXXVI according to the route depicted in scheme H): 
The BOC-protected amino acids required in Scheme A, step 2, can be obtained according to the following procedure (Scheme I): 
HIV Protease Inhibition Assay
HIV protease inhibitory activity was assessed using an adaptation of the method of Matayoshi. et al. [Matayoshi E. D. et al (1990). Science. 247. 954-958]
Crude HIV-1 protease was prepared from E. coli pPTxcex94N. Cultures were grown at 30xc2x0 C. in M9 medium supplemented with 0.2% casamino acids, 100 xcexcg/ml ampicillin and 25 xcexcg/ml thiamine until OD600=0.5-0.6, and the temperature was raised to 42xc2x0 C. to induce expression of the protease. After 1.5 hours, the cells were harvested and the pellets stored at xe2x88x9270xc2x0 C. until required.
The protease was prepared by lysis of the cells in a French pressure cell followed by precipitation of the enzyme with ammonium sulfate at 30% saturation.
The assay was based on intramolecular fluorescence energy transfer using a quenched fluorogenic substrate DABCYL-Ser.Gln.Asn.Tyr.Pro.Ile.Val.Gln.-EDANS, the peptide sequence of which was derived from one of the natural polypeptide processing sites of HIV-1 protease.
The peptide substrate was dissolved in spectroscopic grade dimethyl sulphoxide (DMSO) to give a stock solution of 500 xcexcM. Inhibitors were dissolved in a 1:9 mixture of DMSO:0.1% aqueous Tween 20 to give inhibitor concentrations 20xc3x97 more concentrated than the desired final concentration. The assay buffer comprised 0.1M sodium acetate pH 4.7, 8 mM EDTA, 0.2 M NaCl.
10 xcexcl HIV-1 protease diluted in a 1:1 mixture of 0.1% Tween: assay buffer (concentration adjusted to give approximately 20% substrate turnover) was added to a mixture comprising 455 xcexcl assay buffer, 25 xcexcl inhibitor solution, 10 xcexcl substrate solution. Tubes were incubated for 2 hours at 37xc2x0 C. and the reaction was terminated by the addition of 500 xcexcl of a 2:1 mixture of DMSO: 50 mM Tricine pH 8.5. Fluorescence was measured in a fluorescence spectrophotometer, excitation xcex=340 nm, emission xcex=492 nm.
Antiviral Assay Method
Anti-HIV antiviral activity was assessed using an adaptation of the method of Pauwels et al. [Pauwels et al., 1988, J. Virol. Methods 20: 309-321]. The method is based on the ability of compounds to protect HIV-infected T lymphoblastoid cells (MT4 cells) from cell-death mediated by the infection. The endpoint of the assay was calculated as the concentration of compound at which the cell viability of the culture was preserved by 50% (xe2x80x9850% inhibitory concentrationxe2x80x99, IC50). The cell viability of a culture was determined by the uptake of soluble, yellow 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and its reduction to a purple insoluble formazan salt. After solubilization, spectrophotometric methods were employed to measure the amount of formazan product. MT4 cells were prepared to be in logarithmic-phase growth and a total of 2xc3x97106 cells infected with either the wild type or site directed mutant clones of HIV-HXB2 at a multiplicity of approximately 0.0001 infectious units of virus per cell in a total volume of between 200-500 xcexcl. The cells were incubated with virus for one hour at 37xc2x0 C. then washed in 0.01 M phosphate buffered saline, pH 7.2, and resuspensed in culture medium for incubation in culture with serial dilutions of test compound. The culture medium used was RPMI 1640 without phenol red, supplemented with penicillin, streptomycin, L-glutamine and 10% fetal calf serum (GM 10).
Test compounds were prepared as 2 mM solutions in dimethyl sulphoxide (DMSO). Four replicate, serial 2-fold dilutions in GM10 were then prepared and 50 microlitre amounts placed in 96-well plates over a final concentration range of 625-1.22 nm. Fifty microlitres GM10 and 3.75xc3x97104 infected cells were then added to each well. Control cultures containing no cells (blank), uninfected cells (100% viability; 4 replicates) and infected cells without compound (total virus-mediated cell death; 4 replicates) were also prepared. The cultures were then incubated at 37xc2x0 C. in a humidified atmosphere of 5% CO2 in air for 5 days.
A fresh solution of 5 mg/mL MTT was prepared in 0.01 M phosphate buffered saline, pH 7.2 and 20 xcexcL added to each culture. The cultures were further incubated as before for 2 hours. They were then mixed by pipetting up and down, and 170 microlitres of Triton X-100 in acidified isopropanol (10% v/v Triton X-100 in 1:250 mixture of concentrated HCl in isopropanol) were added and the cultures were mixed again by pipetting up and down. When the formazan deposit was fully solubilised by further mixing, the absorbance (OD) of the cultures was measured at 540 nm and 690 nm wavelength (690 nm readings were used as blanks for artefacts between wells). The percent protection for each treated culture was then calculated from the equation:       %    ⁢          xe2x80x83        ⁢    Protection    =                                                                        (                                  OD                  ⁢                                      xe2x80x83                                    ⁢                  drug                  ⁢                                      -                                    ⁢                  treated                  ⁢                                      xe2x80x83                                    ⁢                  cultures                                )                            -                                                                          (                              OD                ⁢                                  xe2x80x83                                ⁢                untreated                ⁢                                  xe2x80x83                                ⁢                virus                ⁢                                  xe2x80x83                                ⁢                control                ⁢                                  xe2x80x83                                ⁢                cultures                            )                                                                                                      (                                  OD                  ⁢                                      xe2x80x83                                    ⁢                  uninfected                  ⁢                                      xe2x80x83                                    ⁢                  cultures                                )                            -                                                                          (                              OD                ⁢                                  xe2x80x83                                ⁢                untreated                ⁢                                  xe2x80x83                                ⁢                virus                ⁢                                  xe2x80x83                                ⁢                control                ⁢                                  xe2x80x83                                ⁢                cultures                            )                                            xc3x97    100    ⁢          xe2x80x83        ⁢    %  
The IC50 was then obtained from graph plots of percentage protection versus log10 drug concentration.
The IC50 of the compounds of the present invention is as a rule up to 10,000 nM, preferably up to 60 nM, and most preferably up to 10 nM.
IC50 values for selected compounds are shown in the following table:
The compounds of the present invention, as well as their pharmaceutically usable acid addition salts, can be used as medicaments, e.g. in the form of pharmaceutical preparations. The pharmaceutical preparations can be administered orally, e.g. in the form of tablets, coated tablets, dragxc3xa8es, hard and soft gelatin capsules, solutions, emulsions or suspensions. The administration can, however, also be effected rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of injection solutions.
The compounds of the present invention and their pharmaceutically usable acid addition salts can be processed with pharmaceutically inert, inorganic or organic excipients for the production of tablets, coated tablets, dragees and hard gelatin capsules. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts etc. can be used as such excipients e.g. for tablets, dragees and hard gelatine capsules.
Suitable excipients for soft gelatine capsules are e.g. vegetable oils, waxes, fats, semi-solid and liquid polyols etc.
Suitable excipients for the manufacture of solutions and syrups are e.g. water, polyols, saccharose, invert sugar, glucose etc.
Suitable excipients for injection solutions are e.g. water, alcohols, polyols, glycerol, vegetable oils etc.
Suitable excipients for suppositories are e.g. natural or hardened oils, waxes, fats, semi-liquid or liquid polyols etc.
Moreover, the pharmaceutical preparations can contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances.
The dosage can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, in the case of oral administration a daily dosage of about 10 to 2500 mg per person of a compound of formula I should be appropriate, although the above upper limit can also be exceeded when necessary.
The daily dosage can be administered as a single dosage or in divided dosages. The treatment may be in conjunction with the administration of one or more additional therapeutically active substance(s), and such administration may be concurrent or sequential with respect to that of the compounds of formula I. Thus, concurrent administration, as used herein, includes administration of the agents in conjunction or combination, together, or before or after each other.

To an ice-cooled solution of 4-benzyl-5-(2-tert-butylcarbamoyl-4-methoxy-cyclohexylmethyl)-2,2-dimethyl-oxazolidine-3-carbocylic acid tert-butyl ester (358 mg, 0.69 mmol) in dichloromethane (3 ml) and trifluoroacetic acid (3 ml), three drops of water were added. The resulting mixture was stirred at 0xc2x0 C. for 1 hour and then concentrated in vacuo. The residue was co-evaporated twice with toluene, re-dissolved in dichloromethane, washed with a 5% aqueous sodium hydrogen carbonate solution and brine, dried (anhydrous magnesium sulfate) and concentrated in vacuo to give 2-(3-amino-2-hydroxy-4-phenyl-butyl)-5-methoxy-cyclohexanecarboxylic acid tert-butylamine as a white foam (200 mg) which was used in the next step without further purification.
In an analogous manner, the following compounds have been made:

An ice-cooled mixture of 2-(3-amino-2-hydroxy-4-phenyl-butyl)-5-methoxy-cyclohexanecarboxylic acid tert-butylamine (93 mg, 0.247 mmol) and 2-tert-butoxycarbonylamino-3-(naphthalen-2-ylsulfanyl)-propionic acid (95 mg, 0.27 mmol) in dichloromethane (2 ml) and N,Nxe2x80x2-dimethylformamide (1 ml) was treated sequentially with N-ethyl morpholine (144 xcexcl, 1 mmol),1-hydroxybenzotriazole hydrate (57 mg, 0.31 mmol) and N-ethyl-Nxe2x80x2-(3-dimethylaminopropyl)carbodiimide hydrochloride (63 mg, 0.30 mmol). The resulting mixture was allowed to warm to room temperature, stirred overnight and finally concentrated in vacuo. The residue was dissolved in ethyl acetate, washed sequentially with 5% aqueous sodium hydrogen carbonate solution, water, 2N hydrochloric acid and water. The organic phase was dried (anhydrous magnesium sulfate) and concentrated in vacuo to give, after purification by flash chromatography on silica gel (dichloromethane/methanol: 98/2), [1-[1-benzyl-3-(2-tert-butylcarbamoyl-4-methoxy-cyclohexyl)-2-hydroxy-propylcarbamoyl]-2-(naphthalen-2-ylsulfanyl)-ethyl]-carbamic acid tert-butyl ester (136 mg, 78%) as a white foam.
The following compounds were prepared in a similar manner by coupling the appropriate acids and amines: